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The phenotype of the rotavirus SA-11 mutant tsB carrying a thermosensitive mutation in gene 3, which encodes VP3, was characterized further from both infected cells and purified viral particles. The mutant phenotype was initially identified as negative for in vivo double- and single-stranded RNA synthesis. Our results show that the in vitro transcriptional properties of the tsB mutant at the restrictive temperature were identical to those of the wild-type strain. Similar results were obtained with respect to the VP3-associated guanylyl-transferase activity. Analysis of viral particles made by mutant-infected cells at the restrictive temperature showed that only empty single-shelled particles were assembled. This indicates that viral morphogenesis is halted after the initial viral transcription and before RNA replication, suggesting that VP3 may be required as part of the replicase system but not for subviral particle assembly. These data suggest that such a phenotype is not due to alteration of a VP3 function related to transcription.[1]